This invention relates to methods of enhancing resident populations of microorganisms or suppressing undesirable populations of microorganisms at selected sites of the gastrointestinal tract of animals including humans. As used in this specification, probiotic or probiotic microorganism is a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance. This is the definition provided by R. Fuller (AFRC Institute of Food Research, Reading Laboratory, UK) inxe2x80x94Journal of Applied Bacteriology, 1989. 66, pp.365-378. xe2x80x9cProbiotics in Man and Animalsxe2x80x94A Reviewxe2x80x9d, and has subsequently been extended to include supplements and food for humans.
The gastrointestinal tract microflora of the healthy subject protects the host from pathogen invasion. In the young, the elderly and the compromised patient, however, this protective barrier is less effective. An individual can be compromised to various degrees ranging from minor stress and related events, for example, dietary changes, emotional and nutritional stresses, to extreme cases such as in immuno-compromised patients and patients undergoing radio- and chemotherapy.
Probiotic bacteria have been described to exert antimicrobial effects which refers to the actions of the probiotic preparation on another microbe or group of microbes in the gastrointestinal tract. These are directly applicable to the use of probiotics for enhanced resistance against intestinal pathogens, prevention of diarrhoea and constipation. The types of interactions include competitive colonisation as well as adhesion and growth inhibition.
Competitive colonisation refers to the fact that the probiotic strain can successfully out-compete the pathogen for either nutrients or the site of colonisation. Since many gastrointestinal pathogens attach to the intestinal mucosa as the first step in infection, it would be beneficial to the host if this adhesion could be inhibited. There are reports that lactobacilli produce components which inhibit attachment of enterotoxigenic Escherichia coli to intestinal mucosa. In addition, various compounds produced during growth of the probiotic have been shown to inhibit pathogen growth. These include organic acids such as lactic and acetic acid, reuterin and bacteriocins. Organic acids lower the pH and thereby can indirectly affect growth of the pathogen. In addition, the lactic and acetic acids can be toxic to microbes. Reuterin which inhibits the growth of a very broad range of cells is produced by Lactobacillus reuteri when grown in the presence of glycerol. Numerous bacteriocins have been reported to be produced by lactobacilli e.g. Acidophilin, Acidolin, Lactocidin, Bacteriocin, Bulgarican, Lactolin, Lactobacillin and Lactobrevin. They can either have a very broad range of activity or alternatively specifically inhibit the growth of a very limited range of closely related microbes. For example, Lactobacillus sp can exhibit specific antagonistic effects towards Clostridium ramnosum. 
There are different levels of specific bacterial populations in the various regions of the gastrointestinal tract of humans and animals. In addition, it has been shown that the specific strains of the various genera and species vary from one region of the digestive tract to another. It has been shown that dietary fibre influences microbial activity and gas production in the various regions of the gastrointestinal tract of pigs.
In humans it is known that the major carbohydrate sources for bacterial growth in the colon are provided by dietary and endogenous means and that bacteria in the proximal colon have a relatively high supply of dietary nutrients and grow at a fast rate causing a decrease in nutrients available in the distal region resulting in bacteria growing more slowly and the pH frequently approaches neutrality. Because of these varying physiochemical conditions, gross metabolic differences are likely to occur between bacteria resident in the right or left sides of the colon. There is a correlation between the fast and slow rate of bacterial growth in the proximal and distal colon, respectively, with the incidence of disease, including cancer. In the region of fast growth, there is a lower incidence of disease than in the distal colon.
It is the contention of many scientists that the health and well being of people can be positively or negatively influenced by the microorganisms which inhabit the gastrointestinal tract, and in particular, the large bowel. These microorganisms through the production of toxins, metabolic by-products, short chain fatty acids, and the like affect the physiological condition of the host.
The constitution and quantity of the gut microflora can be influenced by conditions or stress induced by disease, life style, travel, and other factors. If microorganisms which positively affect the health and well being of the individual can be encouraged to populate the large bowel, this should improve the physiological well being of the host.
The introduction of beneficial microorganisms, or probiotics, is normally accomplished by the ingestion of the organisms in drinks, yoghurts, capsules, and other forms in such a way that the organism arrives in a viable condition in the large bowel.
It has been demonstrated by Englyst H. N. et al (1987) xe2x80x9cPolysaccharides breakdown by mixed populations of human faecal bacteriaxe2x80x9d, FEMS Microbiology Ecol 95: 163-71, that the bacterial fermentation of resistant starch in the large bowel produces elevated levels of short chain fatty acids, particularly beneficial types such as propionate and butyrate.
The present inventors have realised that it would be desirable to not only deliver probiotic microorganisms to the large bowel but also to provide a medium that would function to promote the growth of the microorganisms when they reach the large bowel.
Surprisingly, it has been found that modified or unmodified resistant starches may function both as a means to transport the probiotic microorganisms to the large bowel and as a growth medium for the microorganism delivered to the target region of the large bowel. It has also been shown in International publication number WO 96/08261, the content of which is incorporated into this specification for the purposes of convenient cross-reference, that resistant starch may be eroded or pitted to afford protection of the associated probiotic microorganisms and that the microorganisms may also adhere to these starch granules. There is a need, however, to be able to deliver probiotics in a more efficient and economical manner.
It would also be desirable to be able to deliver substrate to specific sites of the gastrointestinal tract so as to either enhance or suppress the growth of particular populations of microorganisms at those sites without substantially affecting the populations of other microorganisms at other sites. The present inventors have developed improved methods for altering or influencing microbial populations of the gastrointestinal tract of animals including humans.
In a first aspect, the present invention consists in a method of enhancing a resident population of microorganism in a selected site of the gastrointestinal tract of an animal, the method comprising providing to the animal a selected modified or unmodified resistant starch or mixtures thereof in combination with one or more probiotic microorganisms such that upon ingestion the starch passes through the gastrointestinal tract substantially unutilized until it reaches the selected site where it is utilised by the resident and/or the probiotic microorganisms thereof causing an increase in number and/or activity of the microorganisms.
In a second aspect, the present invention consists in a method of suppressing an undesired resident population of microorganism in a selected site of the gastrointestinal tract of an animal, the method comprising providing to the animal a modified or unmodified resistant starch or mixtures thereof in combination with one or more probiotic microorganisms such that upon ingestion the starch passes through the gastrointestinal tract substantially unutilized until it reaches the selected site where it is utilised by another resident and/or the probiotic microorganisms causing an increase in number and/or activity of the other microorganisms and suppressing the growth and/or activity of the undesired microorganism.
By selecting a resistant starch or a specific modification of resistant starch in combination with a probiotic preparation of one or more microorganisms, it is possible to deliver substrates which are more poorly used by the microorganisms of one part of the colon than another part. For example, the microorganisms in the proximal colon may poorly utilise the resistant starch selected than those microorganisms in the distal colon. Similarly, it is possible to cause one population of microorganism at a specific site of the gastrointestinal tract to grow while the remaining resident populations remain static or are suppressed by the increased growth or activity of the selected population and/or the probiotic microorganisms.
The present invention can also be used to promote growth of desirable probiotic and/or indigenous microbes in the small intestine or stomach where the levels of indigenous organisms are lower and pathogens frequently establish e.g. H. pylori in the stomach or enterotoxigenic Escherichia coli in the small intestine.
In a third aspect, the present invention consists in a method of suppressing a microbial pathogen in the gastrointestinal tract of an animal comprising administering to the animal one or more probiotic microorganisms and a carrier which will function to transport the one or more probiotic microorganisms to the large bowel or other regions of the gastrointestinal tract, the carrier comprising a modified or unmodified resistant starch or mixtures thereof, which carrier acts as a growth or maintenance medium for the non-pathogenic microorganisms in the large bowel or other regions of the gastrointestinal tract to an extent sufficient to suppress growth and/or activity of the microbial pathogen.
In a fourth aspect, the present invention consists in an improved probiotic composition comprising one or more probiotic microorganisms and a carrier which will function to transport the one or more probiotic microorganisms to the large bowel or other regions of the gastrointestinal tract, the carrier comprising modified or unmodified resistant starch or mixtures thereof to which the probiotic microorganisms are bound in a manner so as to protect the microorganisms during passage to the large bowel or other regions of the gastrointestinal tract, which carrier acts as a growth or maintenance medium for microorganisms in the large bowel or other regions of the gastrointestinal tract.
In a fifth aspect, the present invention is directed to an improved method of providing probiotic microorganisms to the gastrointestinal tract of an animal, the improved method comprising administering to the animal one or more probiotic microorganisms and a carrier which will function to transport the one or more probiotic microorganisms to the large bowel or other regions of the gastrointestinal tract, the carrier comprising modified or unmodified resistant starch or mixtures thereof to which the probiotic microorganisms are bound in a manner so as to protect the microorganisms during passage to the large bowel or other regions of the gastrointestinal tract, which carrier acts as a growth or maintenance medium for microorganisms in the large bowel or other regions of the gastrointestinal tract.
In a preferred form, the probiotic microorganisms are bound irreversibly to the modified or unmodified resistant starch.
In a sixth aspect, the present invention consists in a method of reducing the incidence of colorectal cancer or colonic atrophy in an animal, the method comprising providing to the animal one or more SCFA producing probiotic microorganisms and a carrier which will function to transport the one or more probiotic microorganisms to the large bowel or other regions of the gastrointestinal tract, the carrier comprising a modified or unmodified resistant starch or mixtures thereof, which carrier acts as a growth or maintenance medium for microorganisms in the large bowel or other regions of the gastrointestinal tract so as to enhance SCFA production by probiotic and/or resident microorganisms in the gastrointestinal tract of the animal.
In a preferred form of the present invention, the SCFA is butyrate and the microorganisms in the gastrointestinal tract are Cl. butyricum and/or Eubacterium. In order to further enhance the levels of SCFA, the probiotic composition includes Cl. butyricum and/or Eubacterium.
It will be appreciated that the modified or unmodified resistant starch or mixtures thereof may also act as a growth or maintenance medium for microorganisms in the large bowel or other regions of the gastrointestinal tract so as to enhance short chain fatty acid (SCFA) production by microorganisms in the gastrointestinal tract of the animal.
As used in this specification, xe2x80x9cresistant starchxe2x80x9d includes those forms defined as RS1, RS2, RS3 and RS4 as defined in Brown, McNaught and Moloney (1995) Food Australia 47: 272-275. Either modified or unmodified resistant starches or mixtures thereof are used in this invention. The advantage of selected resistant starches is that they are not digested until they reach the selected site of gastrointestinal tract. Therefore, when used in combination with a probiotic, they also provide a readily available substrate for fermentation by the probiotic microorganisms as soon as they arrive in the selected site of the gastrointestinal tract. A preferred form of resistant starch is a high amylose starch particularly high amylose starches as disclosed and taught in WO 94/03049 and WO 94/14342, the contents of which are incorporated into this specification for the purposes of convenient cross-reference.
In WO 94/03049 and WO 94/14342, high amylose starches are disclosed which are resistant starches and include maize starch having an amylose content of 50% w/w or more, particularly 80% w/w or more, rice and wheat starch having an amylose content of 27% w/w or more and: particular granular size ranges of starches having an amylose content of 50% or more and enhanced resistant starch content, these starches including maize. barley, wheat and legumes. This invention is not, however, limited to use of these forms of resistant starch. For example, other forms of resistant starch are derived from sources such as bananas, fruits and potatoes.
It may be advantageous to also chemically modify the starch to, for instance, alter the charge density or hydrophobicity of the granule and/or granule surface to enhance the attachment compatibility between the microorganism and the resistant starch. Chemical modifications, such as etherification, esterification, acidification and the like are well known in this art as being suitable chemical treatments. Similarly other modifications can be induced physically, enzymically or by other means known to the art.
It may also be useful to modify the degree of enzyme susceptibility of the resistant starch by altering the conformation or structure of the starch. Examples include acid or enzyme thinning and cross bonding using difunctional reagents.
One useful modification is the amylolysis of high amylose starches to give starch granules characterised by pits or erosions which can extend from the surface to the interior of the granules. These pits allow the entry of enzymes to the more enzyme susceptible core of the starch granule which is solubilised.
As used herein, Hi-maize(trademark) (trade mark) refers to a high amylose starch obtained from Starch Australasia Limited.
In order that the present invention may be more clearly understood, preferred forms thereof will be described with reference to the following figures and examples.